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Mutational analysis of the Notch2 negative regulatory region identifies key structural elements for mechanical stability.

Stephenson NL, Avis JM - FEBS Open Bio (2015)

Bottom Line: Here, mutations are made within the heterodimerization (HD) domain of the NRR that are known to cause constitutive activation of Notch1 whilst having no effect on the chemical stability of Notch2.Comparison of the mechanical stability and simulated forced unfolding of recombinant Notch2 NRR proteins demonstrates a reduced stability following mutation and identifies two critical structural elements of the NRR in its response to force - the linker region between Lin12-Notch repeats LNRA and LNRB and the α3 helix within the HD domain - both of which mask the S2 cleavage site prior to Notch activation.In two mutated proteins, the LNRC:HD domain interaction is also reduced in stability.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.

ABSTRACT
The Notch signalling pathway is fundamental to cell differentiation in developing and self-renewing tissues. Notch is activated upon ligand-induced conformational change of the Notch negative regulatory region (NRR), unmasking a key proteolytic site (S2) and facilitating downstream events. The favoured model requires endocytosis of a tightly bound ligand to transmit force to the NRR region, sufficient to cause a structural change that exposes the S2 site. We have previously shown, using atomic force microscopy and molecular dynamics simulations, that application of force to the N-terminus of the Notch2 NRR facilitates metalloprotease cleavage at an early stage in the unfolding process. Here, mutations are made within the heterodimerization (HD) domain of the NRR that are known to cause constitutive activation of Notch1 whilst having no effect on the chemical stability of Notch2. Comparison of the mechanical stability and simulated forced unfolding of recombinant Notch2 NRR proteins demonstrates a reduced stability following mutation and identifies two critical structural elements of the NRR in its response to force - the linker region between Lin12-Notch repeats LNRA and LNRB and the α3 helix within the HD domain - both of which mask the S2 cleavage site prior to Notch activation. In two mutated proteins, the LNRC:HD domain interaction is also reduced in stability. The observed changes to mechanical stability following these HD domain mutations highlight key regions of the Notch2 NRR that are important for mechanical, but not chemical, stability. This research could also help determine the fundamental differences in the NRRs of Notch1 and Notch2.

No MeSH data available.


Comparison of the unfolding forces and extensions when wild type and variant hN2-NRR constructs are exposed to AFM unfolding. Frequency of force (A) and extension (B) events occurring during unfolding features in the wild type construct (bar graph) compared to A1647P, L1573P and V1623D (various lines, as shown in key) highlighting a major reduction in force with little change to the extensions observed. A bimodal distribution of the force frequencies for wild type is highlighted by grey lines.
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f0020: Comparison of the unfolding forces and extensions when wild type and variant hN2-NRR constructs are exposed to AFM unfolding. Frequency of force (A) and extension (B) events occurring during unfolding features in the wild type construct (bar graph) compared to A1647P, L1573P and V1623D (various lines, as shown in key) highlighting a major reduction in force with little change to the extensions observed. A bimodal distribution of the force frequencies for wild type is highlighted by grey lines.

Mentions: When these simulation results are compared to those observed within the AFM experiments a similar pattern is observed. Example raw AFM force-extension profiles for mechanical unfolding of the WT and mutated hN2-NRR constructs are shown in Fig. 3. It is clear from these raw profiles that the force required for unfolding of the NRR following A1647P, L1573P and V1623D mutations is reduced. Within the A1647P construct this appears to be confined to the LNR unfolding force (first cluster of 3 peaks, Fig. 3), whilst the L1573P and V1623D mutations cause a reduction in the force required for unfolding across the whole construct compared to the WT profile. These data confirm trends observed within the MD simulations, suggesting a destabilisation within these constructs leading to lower forces required for unfolding the LNRA and B regions. Furthermore, analysis of collected sets of such forced unfolding data gained through AFM experiments shows the same three mutations (A1647P, L1573P and V1623D) reduce the force required for unfolding of the NRR compared to the wild type (Fig. 4). Whereas, introducing mutations I1627N and F1565S showed little change (Fig. S1). Unfolding of WT hN2-NRR shows a bimodal distribution (median = 179 and 373 pN). The initial peak in the frequency of the force data has previously been identified as corresponding to the unfolding of LNR modules A and B, whereas the broader distributed peak, at around 350 pN, was identified as HD domain unfolding, possibly combined with LNRC (the broad distribution attributed to non-specific interactions between Lys side chains and the AFM slide during the attachment process [18]). L1573P and V1623D show the greatest reductions in force, lowering the mean force of unfolding from 341.8 to 177.1 pN (L1573P) and 147.4 pN (V1623D). A1647P shows an increase in the number of unfolding events at the lower force of 150 pN as well as a slight reduction in the number of unfolding events requiring higher forces (mean force of unfolding 247.3 pN). These changes in the force required for unfolding between the WT and mutated constructs were all found to be highly statistically significant (P < 0.001). This further confirms results observed within the MD simulations indicating a reduction in the force required for unfolding resulting from mutating residues A1647, L1563 and V1623. Furthermore, data suggests this reduction in force is related to the unfolding of the LNR domains, and from the MD simulations can be more specifically located to the removal of the inhibitory plug formed by residues in the LNRA:B linker region and LNRB, key events in removing the auto inhibition and allowing for cleavage at the S2 site. Simulations of the other three mutations (F1565S, L1566P and I1627N) show some reduction in force required for the LNRB unfolding, whilst maintaining the force required for LNRA:B linker removal (Fig. 2). This could also suggest a mechanism by which these mutations could affect the forced unfolding of the NRR, though the effects are less clear in these mutants. Furthermore, AFM for these mutated constructs shows much smaller reductions in the force required for unfolding (Figs. 7 and S1).


Mutational analysis of the Notch2 negative regulatory region identifies key structural elements for mechanical stability.

Stephenson NL, Avis JM - FEBS Open Bio (2015)

Comparison of the unfolding forces and extensions when wild type and variant hN2-NRR constructs are exposed to AFM unfolding. Frequency of force (A) and extension (B) events occurring during unfolding features in the wild type construct (bar graph) compared to A1647P, L1573P and V1623D (various lines, as shown in key) highlighting a major reduction in force with little change to the extensions observed. A bimodal distribution of the force frequencies for wild type is highlighted by grey lines.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4537882&req=5

f0020: Comparison of the unfolding forces and extensions when wild type and variant hN2-NRR constructs are exposed to AFM unfolding. Frequency of force (A) and extension (B) events occurring during unfolding features in the wild type construct (bar graph) compared to A1647P, L1573P and V1623D (various lines, as shown in key) highlighting a major reduction in force with little change to the extensions observed. A bimodal distribution of the force frequencies for wild type is highlighted by grey lines.
Mentions: When these simulation results are compared to those observed within the AFM experiments a similar pattern is observed. Example raw AFM force-extension profiles for mechanical unfolding of the WT and mutated hN2-NRR constructs are shown in Fig. 3. It is clear from these raw profiles that the force required for unfolding of the NRR following A1647P, L1573P and V1623D mutations is reduced. Within the A1647P construct this appears to be confined to the LNR unfolding force (first cluster of 3 peaks, Fig. 3), whilst the L1573P and V1623D mutations cause a reduction in the force required for unfolding across the whole construct compared to the WT profile. These data confirm trends observed within the MD simulations, suggesting a destabilisation within these constructs leading to lower forces required for unfolding the LNRA and B regions. Furthermore, analysis of collected sets of such forced unfolding data gained through AFM experiments shows the same three mutations (A1647P, L1573P and V1623D) reduce the force required for unfolding of the NRR compared to the wild type (Fig. 4). Whereas, introducing mutations I1627N and F1565S showed little change (Fig. S1). Unfolding of WT hN2-NRR shows a bimodal distribution (median = 179 and 373 pN). The initial peak in the frequency of the force data has previously been identified as corresponding to the unfolding of LNR modules A and B, whereas the broader distributed peak, at around 350 pN, was identified as HD domain unfolding, possibly combined with LNRC (the broad distribution attributed to non-specific interactions between Lys side chains and the AFM slide during the attachment process [18]). L1573P and V1623D show the greatest reductions in force, lowering the mean force of unfolding from 341.8 to 177.1 pN (L1573P) and 147.4 pN (V1623D). A1647P shows an increase in the number of unfolding events at the lower force of 150 pN as well as a slight reduction in the number of unfolding events requiring higher forces (mean force of unfolding 247.3 pN). These changes in the force required for unfolding between the WT and mutated constructs were all found to be highly statistically significant (P < 0.001). This further confirms results observed within the MD simulations indicating a reduction in the force required for unfolding resulting from mutating residues A1647, L1563 and V1623. Furthermore, data suggests this reduction in force is related to the unfolding of the LNR domains, and from the MD simulations can be more specifically located to the removal of the inhibitory plug formed by residues in the LNRA:B linker region and LNRB, key events in removing the auto inhibition and allowing for cleavage at the S2 site. Simulations of the other three mutations (F1565S, L1566P and I1627N) show some reduction in force required for the LNRB unfolding, whilst maintaining the force required for LNRA:B linker removal (Fig. 2). This could also suggest a mechanism by which these mutations could affect the forced unfolding of the NRR, though the effects are less clear in these mutants. Furthermore, AFM for these mutated constructs shows much smaller reductions in the force required for unfolding (Figs. 7 and S1).

Bottom Line: Here, mutations are made within the heterodimerization (HD) domain of the NRR that are known to cause constitutive activation of Notch1 whilst having no effect on the chemical stability of Notch2.Comparison of the mechanical stability and simulated forced unfolding of recombinant Notch2 NRR proteins demonstrates a reduced stability following mutation and identifies two critical structural elements of the NRR in its response to force - the linker region between Lin12-Notch repeats LNRA and LNRB and the α3 helix within the HD domain - both of which mask the S2 cleavage site prior to Notch activation.In two mutated proteins, the LNRC:HD domain interaction is also reduced in stability.

View Article: PubMed Central - PubMed

Affiliation: Faculty of Life Sciences, Manchester Institute of Biotechnology, University of Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom.

ABSTRACT
The Notch signalling pathway is fundamental to cell differentiation in developing and self-renewing tissues. Notch is activated upon ligand-induced conformational change of the Notch negative regulatory region (NRR), unmasking a key proteolytic site (S2) and facilitating downstream events. The favoured model requires endocytosis of a tightly bound ligand to transmit force to the NRR region, sufficient to cause a structural change that exposes the S2 site. We have previously shown, using atomic force microscopy and molecular dynamics simulations, that application of force to the N-terminus of the Notch2 NRR facilitates metalloprotease cleavage at an early stage in the unfolding process. Here, mutations are made within the heterodimerization (HD) domain of the NRR that are known to cause constitutive activation of Notch1 whilst having no effect on the chemical stability of Notch2. Comparison of the mechanical stability and simulated forced unfolding of recombinant Notch2 NRR proteins demonstrates a reduced stability following mutation and identifies two critical structural elements of the NRR in its response to force - the linker region between Lin12-Notch repeats LNRA and LNRB and the α3 helix within the HD domain - both of which mask the S2 cleavage site prior to Notch activation. In two mutated proteins, the LNRC:HD domain interaction is also reduced in stability. The observed changes to mechanical stability following these HD domain mutations highlight key regions of the Notch2 NRR that are important for mechanical, but not chemical, stability. This research could also help determine the fundamental differences in the NRRs of Notch1 and Notch2.

No MeSH data available.